MLL-rearranged acute lymphoblastic leukaemia stem cell interactions with bone marrow stroma promote survival and therapeutic resistance that can be overcome with CXCR4 antagonism

Abstract

Infants with MLL-rearranged (MLL-R) acute lymphoblastic leukaemia (ALL) have a dismal prognosis. While most patients achieve remission, approximately half of patients recur with a short latency to relapse. This suggests that chemotherapy-resistant leukaemia stem cells (LSCs) survive and can recapitulate the leukaemia. We hypothesized that interactions between LSCs and the bone marrow microenvironment mediate survival and chemotherapy resistance in MLL-R ALL. Using primary samples of infant MLL-R ALL, we studied the influence of bone marrow stroma on apoptosis, proliferation, and cytotoxicity induced by the FLT3 inhibitor lestaurtinib. MLL-R ALL were differentially protected by stroma from spontaneous apoptosis compared to non-MLL-R ALL. Co-culture of bulk MLL-R ALL in direct contact with stroma or with stroma-produced soluble factors promoted proliferation and cell cycle entry. Stroma also protected bulk MLL-R ALL cells and MLL-R ALL LSCs from lestaurtinib-mediated cytotoxicity. Previous studies have demonstrated that CXCR4 mediates bone marrow microenvironment signalling. Using a xenograft model of MLL-R ALL, we demonstrated that CXCR4 inhibition with AMD3100 (plerixafor) led to markedly enhanced efficacy of lestaurtinib. Therefore, the bone marrow microenvironment is a mediator of chemotherapy resistance in MLL-R ALL and targeting leukaemia-stroma interactions with CXCR4 inhibitors may prove useful in this high-risk subtype of paediatric ALL.

Fig. 1

Human bone marrow stromal co-culture enhances the survival of primary patient ALL cells, particularly those with MLL rearrangement. Primary patient leukaemic blasts were cultured for 48 h in medium alone or on bone marrow stromal feeder layers. The surviving fraction, as a percentage of the number of viable cells at baseline, was determined for each sample using the annexin V binding apoptosis assay. (A) All samples (n = 28). Each line joins the results for one patient sample. P-value derived from paired Student’s t test. (B) Comparison of MLL-R (n = 9) vs. non MLL-R (n = 19) cultured in medium. P-value derived from Student’s t test. (C) Comparison of MLL-R (n = 9) vs non MLL-R (n = 19) co-cultured with normal human marrow stroma. P-value derived from Student’s t test.

Fig. 2

Stromal co-culture induces higher proliferation, cell cycle entry, and inhibition of apoptosis of bulk MLL-R ALL cells, especially with direct leukaemia-stroma contact. (A) Schema illustrating in vitro bone marrow stroma co-culture system. Leukaemic samples are cultured in three different conditions: in physical contact with stromal feeder layers (S), with stromal feeder layers but separated by permeable transwell (T) membranes (which expose leukaemia cells to soluble factors produced by stroma but prevent cell-cell interactions), and in culture medium only (M). (B) Proliferation was measured after 24 and 48 h of culture using the MTT assay. Error bars represent standard error of the mean (SEM) of triplicate wells. (C) Flow cytometry analysis of DNA content after PI staining was used to measure cell cycle kinetics after 24 and 48 h of culture. Error bars represent SEM of duplicate fluorescence-activated cell sorting (FACS) tubes. (D) Apoptosis was measured using the AVB assay. Error bars represent SEM of duplicate FACS tubes.

Fig. 3

Human bone marrow stromal co-culture protects primary patient MLL-rearranged ALL cells from the cytotoxic effects of FLT3 inhibition. (A) MTT dose-response curves showing cytotoxic response to lestaurtinib (normalized to untreated controls) for three primary MLL-rearranged ALL patient samples cultured either in medium alone (dashed lines), or on bone marrow stromal feeder layers (solid lines) for 48 h. Error bars represent standard error of the mean (SEM) of triplicate wells. (B) Western blot for phosphorylated and total FLT3 after treatment with or without 50 n mol/l lestaurtinib.

Fig. 4

Direct stromal contact results in higher pro-proliferative effects and inhibition of apoptosis, particularly in the presence of FLT3 inhibition. Primary MLL-R ALL cells were cultured in S, T, M conditions and treated with dose ranges of 0–100 n mol/l lestaurtinib for 24 and 48 h. (A) Proliferation was measured using the MTT assay. Data for 50 n mol/l lestaurtinib are shown. Error bars represent standard error of the mean (SEM) of triplicate wells. (B) Apoptosis was measured using the AVB assay. Error bars represent SEM of duplicate FACS tubes.

Fig. 5

Ex vivo stromal co-culture enhances LSC survival and protects LSC from cytotoxic effects of FLT3 inhibition. (A) Schema of experiment where primary MLL-R ALL cells were cultured in S, T and M condition in the absence or presence of 20 n mol/l lestaurtinib, then injected into sub-lethally irradiated NOD/SCID mice. Ten weeks after injection, mice were sacrificed and assessed for engraftment of human leukaemia cells. (B) S and T conditions significantly enhanced LSC survival ex vivo, both in absence (open data points) and presence (filled data points) of 20 n mol/l lestaurtinib. Engraftment is expressed as percentage of marrow CD45 + cells that were of human origin as determined by flow cytometric analysis. Bars show mean and standard deviation for each cohort.

Fig. 6

In vivo treatment of NOD/SCID mice with G-CSF and AMD3100 sensitizes LSC to FLT3 inhibition. (A) Schema illustrating in vivo treatment of NOD/SCID mice injected with primary MLL-R ALL cells. Cohorts of sub-lethally irradiated NOD/SCID mice were injected with primary leukaemia cells (n = 2 primary samples), then treated weekly from week 3 to week 6 with one of four regimens: vehicle-treated control (C); mobilizers (M), consisting of G-CSF 2·5 μg SC twice daily on days 1 and 2 plus AMD3100 (plerixafor) 5 mg/kg SC once on day 3; lestaurtinib 20 mg/kg SC twice daily on days 3–6 alone (L); or mobilizers plus lestaurtinib (M+L). The mice were sacrificed and assessed for engraftment of human leukaemia cells 10 weeks after injection (expressed as the percentage of bone marrow CD45 + cells that were of human origin, as determined by flow cytometry analysis). (B) Results for Sample 1, (C) results for Sample 2.